The polymerase chain reaction (PCR) is one of the most important technologies for genome analysis. One of the weaknesses of PCR is that primer extension from mismatched primers occurs. Extension from mismatched primers limits allele-specific amplification and detection of mutations and polymorphisms to some extent with homogeneous DNA samples (e.g. for genotyping), but to a greater extent for heterogeneous DNA samples (e.g. for detection of cancer mutations). Another of the weaknesses of PCR is much poorer fidelity than observed during in vivo DNA replication, as reflected in (1) a rather high rate of nucleotide misincorporation, leading to difficulty in using PCR for faithful cloning and (2) the production of multiple bands when di- and trinucleotide repeats are amplified. An order of magnitude improvement in PCR specificity and fidelity could increase accuracy in genotyping and somatic mutation detection and open up new uses for PCR, including the reproducible and faithful cloning of genomic DNA fragments up to several kilobases in length. The present invention provides such an improvement in PCR.
The ligase chain reaction (LCR) and its variations (e.g., oligonucleotide ligation assay (OLA), ligase detection reaction (LDR)) are alternative techniques for genome analysis. A commonly recognized source of spurious background signal in LCR and its variations, as well as in PCR and its variations, is the hybridization of an oligonucleotide such as a probe or a primer, to regions of the nucleic acid not intended to be amplified. Generally, these hybridizations occur because the target sample contains, in addition to the target sequence itself, other sequences with some similarity to the target nucleic acid. Although hybridization of probe or primer to these similar sequences is not as probable as to the target sequence, some hybridization can occur. When such unintended non-specific hybridization occurs, it is possible that sequences other than the targeted sequence will be amplified. If these limitations of PCR and LCR could be reduced or eliminated, the methods would be even more useful than they presently are.